The multiple antigen peptide derivative, Leu8-Lys4-Lys2-Lys-βAla (Leu8-MAP), was synthesized by attaching the carboxyl of leucine to the NH2 termini of a branched lysine core, termed MAP, creating a molecule of about 1900 Da with 8 leucine residues. On a molar basis (independent of the number of leucine substitutions), Leu8-MAP was as effective as leucine in suppressing macroautophagy and proteolysis; moreover, it exhibited the same apparent K(m) (about 0.1 mM). The effect was specific for leucine since Ile8-MAP was inactive. It is of interest, though, that Leu8-MAP did not elicit the multiphasic response typical of leucine but instead evoked the single site inhibition normally seen with leucine plus the co-regulator alanine. Some free leucine was produced from Leu8-MAP during hepatocyte incubations, but the amounts were insufficient to account for the inhibition. Although this degradation created species of Leu-MAP that had lost 1-3 residues of leucine, their inhibitory effectiveness was not diminished. Because the extracellular/intracellular distribution ratio of [3H]-Leu8- MAP was 100:1 or greater, the direct transport of Leu8-MAP across the plasma membrane into the cytosolic compartment can be excluded. Hence, cytosolic concentrations of Leu8-MAP will be at least 100-fold smaller than those of leucine under conditions of comparable proteolytic inhibition. For these and related reasons, effects attributable to the recognition of Leu8-MAP cannot be explained by signals generated within the cytosol. They could, however, be mediated from site(s) on the plasma membrane or within associated vesicles.
Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-βAla
MIOTTO, GIOVANNI;VENERANDO, RINA;MARIN, ORIANO;
1994
Abstract
The multiple antigen peptide derivative, Leu8-Lys4-Lys2-Lys-βAla (Leu8-MAP), was synthesized by attaching the carboxyl of leucine to the NH2 termini of a branched lysine core, termed MAP, creating a molecule of about 1900 Da with 8 leucine residues. On a molar basis (independent of the number of leucine substitutions), Leu8-MAP was as effective as leucine in suppressing macroautophagy and proteolysis; moreover, it exhibited the same apparent K(m) (about 0.1 mM). The effect was specific for leucine since Ile8-MAP was inactive. It is of interest, though, that Leu8-MAP did not elicit the multiphasic response typical of leucine but instead evoked the single site inhibition normally seen with leucine plus the co-regulator alanine. Some free leucine was produced from Leu8-MAP during hepatocyte incubations, but the amounts were insufficient to account for the inhibition. Although this degradation created species of Leu-MAP that had lost 1-3 residues of leucine, their inhibitory effectiveness was not diminished. Because the extracellular/intracellular distribution ratio of [3H]-Leu8- MAP was 100:1 or greater, the direct transport of Leu8-MAP across the plasma membrane into the cytosolic compartment can be excluded. Hence, cytosolic concentrations of Leu8-MAP will be at least 100-fold smaller than those of leucine under conditions of comparable proteolytic inhibition. For these and related reasons, effects attributable to the recognition of Leu8-MAP cannot be explained by signals generated within the cytosol. They could, however, be mediated from site(s) on the plasma membrane or within associated vesicles.File | Dimensione | Formato | |
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Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-beta Ala.pdf
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